Cytopathicity of GCV in HSV-tk Transduced C 6 Glioma Cells 
To determine whether introduction of the HSV-tk gene would render C 6 cells 
susceptible to killing by GCV, ADV-tk was used to transduce C 6 glioma cells in culture. The 
viral thymidine kinase activity in cellular extracts was determined by 3 H-acyclovir 
phosphorylation (Fyfe et al., 1978). The HSV-tk gene was expressed at high levels in 
transduced C 6 cells and saturation was achieved after transduction at an M.O.I. of 1000 
(Fig. 4A). To test whether C 6 glioma cells expressing the HSV-tk gene were susceptible to 
GCV toxicity, the transduced cells were treated with either phosphate buffered saline (PBS) 
or 10 mg of GCV per ml of culture medium (Fig. 4B). An M.O.I. of 300 and above resulted 
in greater than 90% cell death. There was no cytotoxic effect of GCV on cells transduced 
with the control virus (ADV-ligal). 
III. ADENOVIRAL-MEDIATED TREATMENT OF C 6 EXPERIMENTAL GLIOMAS IN NUDE 
MICE 
Regression of C 6 Glioma in Brains of Nude Mice After In Vivo Gene Therapy. 
In view of the GCV susceptibility of HSV-tk-expressing tumor cells in vitro, we tested 
whether direct transduction of C 6 gliomas in vivo followed by GCV treatment would cause 
tumor regression. Athymic nude mice were chosen as host animals so that we could test the 
effect of the ADV-tk/GCV treatment could be tested in the absence of a cellular immune 
response. 
Athymic nude mice, 6-8 weeks old, were anesthetized with avertin (30 ml/kg) and 
placed in a stereotaxic frame. A burr hole was drilled in the skull 1 mm anterior and 2 mm 
lateral to the bregma with a 0.9 mm burr to expose the dura. C 6 tumor cells were harvested 
for injection by treating the cells at 37°C with 0.25% trypsin in 1.0 mM ethylenediamine 
tetra-acetic acid (EDTA) for 5 minutes. The cells were collected in DMEM, washed, and 
resuspended in Hanks' balanced salt solution (HBSS) at a concentration of 1.0 x 10 4 
cells/ml. Cells were counted before and after concentrating and prior to injection with a 
hemacytometer. Following the implantation procedure, the viability of the cell preparation 
was assessed by trypan blue exclusion analysis. Using a microliter syringe (Hamilton, Reno, 
NV) fitted with a 26 gauge needle and connected to the manipulating arm of the stereotaxic 
frame IxlO 4 C 6 glioma cells in 1 ml of Hank's buffered saline were injected over 2.5 min into 
the caudate nucleus at a depth of 3.5 mm from the dura. The needle was left in place for 3 
min and then withdrawn slowly over another min. The gliomas grew rapidly in the caudate 
nucleus, invaded the cerebral cortex and spread through the injection needle tract into the 
subgaleal space of the scalp. Untreated animals survived an average of 23 days after 
tumor cell injection. 
Eight days after tumor cell injection, when the tumors were approximately 0.4 mm 2 in 
size, 3x1 O 0 particles of ADV-tk or ADV-bgal were injected stereotaxically into the tumors. 
The same coordinates were used for the injection of virus except that the needle was placed 
1 mm deeper than the tumor cells and the virus was injected at 6 points, 0.5 mm apart, 
along the needle tract as the needle was withdrawn. The needle was coated with carbon 
particles (<30 mm) to mark the needle tract and to verify co-localization of the virus injection 
with the tumor ADV-tk or ADV-bgal virus (3 x 10 8 viral particles in 3 ml in PBS) was 
injected in a volume of 0.5 ml at each of the 6 positions over 10 min. The needle was left in 
the tissue for an additional 3 min and then was slowly withdrawn. The scalp wound was 
closed with Autoclips (Clay Adams, Parsippany, NJ). 
24 ] 
Recombinant DNA Research, Volume 20 
